The invention relates to the identification of microscopic and submicroscopic targets in medical, industrial, or environmental samples.
Overview
Detecting, enumerating, and identifying low levels of molecular targets is a cornerstone of routine medical, industrial, and environmental diagnostics. For example, samples are analyzed to detect molecules from infectious agents, cancer cells, hormones, manufacturing contaminants, and pollutants.
Although numerous disparate and powerful methods for routine detection of low levels of molecules have been commercialized there are still gaps in the testing repertoire. Some of these gaps became apparent following the bioterror attacks in 2001. For example, there is an unmet need for rapid, ultra-sensitive, cost-effective, and user-friendly tests to screen simultaneously for multiple types of agents at the point of exposure. Both healthcare and industry are hampered by the lack of rapid, sensitive, and easy-to-use diagnostic tests that can be conducted on-site by non-medical personnel.
The discussion that follows focuses on routine medical diagnostics; similar methods are used for industrial and environmental applications.
Immunological Methods
Immunological tests, or immunoassays, are ubiquitous in medical diagnostics. Based on the interaction of antibodies and the corresponding targets (called antigens), immunoassays are used to detect a broad range of molecules ranging in size from small (e.g., a drug of abuse) to large (e.g., an HIV protein). Serological tests are immunological assays that, rather than testing directly for antigens, test for a host immunological response to previous exposure to the antigen—i.e., they test for the presence of host antibodies to the antigen. Numerous immunoassay systems are available ranging from large automated central lab systems to over-the-counter pregnancy tests. The tests cover a broad range of formats including agglutination assays, precipitin assays, enzyme-linked immunoassays, direct fluorescence assays, immuno-histological tests, complement-fixation assays, serological tests, immuno-electrophoretic assays, and rapid “strip” tests (e.g., lateral flow and flow through tests). Immunological tests can be extremely simple and rapid. Thus, many of the most desirable tests, those that can be conducted in a physician's office or at home by the patient, are immunological tests. One drawback of commercialized rapid immunological tests is that they are insensitive.
The need for commercially accessible sensitive immunoassays will increase in coming years. One of the first major payoffs of the human genome and proteome projects will be numerous new markers for diagnosing disease states, such as cancer, cardiovascular disease, and neurological disease. These markers may occur in trace amounts in clinical samples, such as blood, urine, and solid tissues. Commercialized rapid immunoassay tests, which are appropriate for high abundance protein markers (such as PSA, myoglobin, and antibodies, for example), may be inadequate for detecting the substantial fraction of proteins present at low concentrations.
Genetic Methods
Genetic methods are general and powerful tools for detecting and identifying nucleic acid molecules from organisms and viruses. Revolutionary new methods for ultra-sensitively detecting and distinguishing the nucleic acid content of cells and viruses based on nucleic acid amplification have recently been developed. For example, commercial tests can detect the nucleic acids from a small number of sub-microscopic HIV virus particles (e.g., 50 particles/ml). Amplification technologies include the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), Transcription Mediated Amplification (TMA), and rolling circle amplification (RCA).
Nucleic acid amplification-based tests have several drawbacks that have hampered their exploitation in routine clinical and commercial diagnostics. The tests are generally much more expensive than microbial culture tests; require high user expertise; involve demanding sample preparation protocols; generally require a time-consuming amplification step; suffer from vulnerability to contamination and false positives; have enzymatic steps that are sensitive to inhibitors found in many clinical samples and lead to false negatives; and often have reproducibility problems in multiplexed applications. Furthermore, like microbial culture methods these methods are not well-suited to detect important classes of non-nuceic acid targets (e.g., toxins, hormones, drugs-of-abuse, and proteins).
Biochemical, Chemical, and Physical Methods
Other technologies for sensitive detection of microscopic and sub-microscopic targets include flow cytometry, mass spectroscopy, biosensors, absorbance spectroscopy, fluorescence polarization, fluorescence fluctuation spectroscopy, electrophoresis, and chromatography, among many others. Because of the expense and expertise typically required, most of these methods have remained in the province of the research laboratory.
One method that has proved useful in clinical diagnostic laboratories is flow cytometry. Flow cytometric methods are used for quantitatively detecting particular cell types on the basis of the ability to bind labeled probes (e.g., stains, antibodies, or nucleic acids). Individual cells or particles are forced to flow through a narrow channel, one at a time, past a laser beam. Fluorescence emission and size/shape information is gathered by analyzing the spectrum and light scattering caused by the organism/particle. Thousands of individual cells or particles can be analyzed per minute. For example, flow cytometry is used to quantify the population sizes of classes of lymphocytes in patients with AIDS. A highly multiplexed flow cytometric method that can detect and identify non-cellular molecules such as proteins or nucleic acids has been commercialized by Luminex (U.S. Pat. No. 5,981,180). Drawbacks of the flow cytometric method include the expense, required operator skill, inability to analyze large sample volumes (which limits sensitivity).
Biosensor technologies also hold promise for sensitive molecular detection. Biosensors use physical methods to convert a biological event, for example binding of an antibody to an antigen, to a detectable signal. One popular biosensor used for molecular detection uses surface plasmon resonance (Mullett, W. M., et al. (2000). Methods 22: 77-91). Thermo BioStar's optical immunoassay (Schultze, D., et al. (2001). Eur J Clin Microbiol Infect Dis 20: 280-3) uses the principle of optical interference to detect binding of antigens to antibodies. The BARC biosensor technology uses magnetoresistive detection (as used for hard disk storage) of analytes tagged with single magnetic microparticles (Edelstein, R. L., et al., Biosens Bioelectron 14: 805-13, 2000).
Point-of-Care and On-Site Methods
Point-of-care tests allow professionals and consumers to perform diagnostic tests outside of dedicated central laboratories. Point-of-care diagnostics is one of the fastest growing segments in in vitro diagnostics. Typical point-of-care testing locations include the patient's bedside in hospitals; physician office labs; nursing homes; public and private health clinics; college health centers; correctional facilities; emergency vehicles; the workplace and home. Numerous point-of-care tests have been introduced in recent years and some have been extraordinarily successful. Commercialized point-of-care tests fall into several categories, those which test for: (1) metabolites (e.g., glucose and urea), inorganic molecules, drugs, and blood gases; (2) tests for macromolecules and hormones; and (3) tests for disease-causing agents (e.g., bacteria, viruses, parasites).
While very successful for analytes that occur at relatively high concentrations (e.g., blood glucose), developing point-of-care tests for low abundance target molecules has been problematic. This difficulty is largely attributable to combining two mutually antagonistic product requirements: (1) the need for sophisticated technology to meet demanding test specifications including ultra-sensitivity and (2) the need for low cost, user-friendly, and portable tests that can be operated by unskilled operators.
Some commercialized or emerging ultra-sensitive point-of-care tests are adaptations of formats used for less sensitive commercialized tests. For example, Response Biomedical Corp. has commercialized a 10-minute strip test (or lateral flow test) based on its RAMP platform, which uses fluorescently dyed microbead labels (Brooks, D. E., et al. (1999). Clin Chem 45: 1676-1678.). Similarly, Biosite's rapid strip tests detect drugs-of-abuse, cardiac markers, and infectious agents (Landry, M. L., et al. (2001). J Clin Microbiol 39: 1855-8.).
Point-of-care tests based on nucleic acid amplification have been difficult to develop because of their complexity and expense. One system is Cepheid's Smart Cycler system. Another nucleic acid amplification test for B. anthracis has been developed by the Mayo Clinic in collaboration with Roche (Light Cycler; Makino, S. I., et al. (2001). Lett Appl Microbiol 33: 237-40.). These systems carry both the advantages and disadvantages inherent to PCR methods. Amplification methods can deliver impressive analytical sensitivity and can be moderately rapid, for example, the Smart Cycler can deliver results in an hour (Belanger, S. D., et al. (2002). Journal of Clinical Microbiology 40: 1436-40). The analytes are limited to those containing nucleic acid (ruling out tests for protein toxins and small molecules, for example); false negatives can result from sample inhibition; highly multiplexed tests are problematic; the tests require trained personnel; and the tests are expensive.
Biosensor-based point-of-care technologies also hold promise as they can combine impressive technical specifications with low-cost and user-friendliness. For example, Thermo Biostar's OIA technology, which can test for pathogens such as influenza virus, relies on a biosensor with an optically activated surface that changes color upon binding of viral antigens because of changes in optical interference (Rodriguez, W. J., et al. (2002). Pediatr Infect Dis J 21: 193-6.).
Some of the emerging technologies focus on improved multiplexing. For example, Biosite is developing a highly parallel capillary microfluidic immunoassay system. Somalogic's technology uses aptamer arrays to analyze samples for hundreds or even thousands of analytes (Brody, E. N., et al. (1999). Mol Diagn 4: 381-8). Nanogen's NanoChip (Ewalt, K. L., et al. (2001). Anal Biochem 289: 162-72) electronic arrays are also being applied to detecting agents of bioterrorism. While tests that screen for hundreds of analytes are of clear value for drug discovery, it is less clear whether this level of multiplexing will be important for point-of-care tests.
Various new labeling/detection strategies for improving the sensitivity of point-of-care tests are also being directed towards tests for biowarfare agents. Orasure's new quantitative drugs-of-abuse tests use UPT (up-converting phosphor technology) particles (Niedbala, R. S., et al. (2001). Anal Biochem 293: 22-30.). The Navy's BARC technology uses magnetic particles and detectors to sensitively analyze samples for specific analytes (Edelstein, R. L., et al. (2000). Biosens Bioelectron 14: 805-13.). Other new labeling methods including quantum dot nanocrystals (Quantum Dot; Wang, C., et al. (2001). Science 291: 2390-2) and resonance light scattering particles (Genicon; Yguerabide, J., et al. (2001). J Cell Biochem Suppl Suppl: 71-81) could also potentially improve the sensitivity of point-of-care tests.
Unmet Needs for Molecular Diagnostics
There is a critical unmet need for efficient biowarfare agent detection by first-responders at the point-of-exposure and by routine automated scanning of building air and water supply. Similarly, because of the lack of sensitive on-site testing, clinic patients can not be sensitively tested on-site for sexually-transmitted disease pathogens, and post-surgical patients can not be quickly and sensitively screened for blood infection. The need for new technologies to address this gap may become even more acute as new diagnostic markers for diseases (including cancer, infectious disease, and cardiovascular disease) inevitably emerge from the genome and proteome projects.